Tools to control the expression of genes in vivo play an essential role in experimental biology and research on disease processes. Nowhere is this more apparent than in the transparent zebrafish embryo, where expression from transgenes such as those encoding fluorescent proteins allows discrete groups of cells or tissues to be labeled and followed over time, and with appropriate regulators, to be genetically modified or targeted for destruction. Binary transcriptional regulatory systems, such as the Gal4/UAS system of yeast, have revolutionized research in Drosophila by enabling precise temporal and spatial control of gene activation. However, this system has been less effective in zebrafish and other vertebrate models, in large part due to the progressive methylation and silencing of multicopy upstream activation sequences (UAS) needed to promote high levels of gene expression from integrated transgenes. In this project, we will adapt the Q regulatory system of Neurospora crassa for use in transgenic zebrafish. The Q system has many advantages, including 1. a higher level of transcriptional activation than achieved by Gal4, 2. a transcriptionl regulator, QF, that can be inactivated by a repressor, QS, 3, the ability to block repression and restore QF activity in the presence of quinic acid and, importantly, 4. a QF binding site (QUAS) that does not contain essential CpG dinucleotides, which are prone to DNA methylation and transcriptional silencing in zebrafish. Recently, the Q system was successfully applied to invertebrate models. Our preliminary data indicate that it also functions effectively to regulate transcription in zebrafish embryos. We propose to validate further the components of the Q system in zebrafish, to develop new methods for intersectional gene expression, to generate all reagents in Gateway compatible vectors for ease of use by other researchers, and to perform a large-scale, enhancer trap screen. We aim to establish a collection QF driver lines that activate reporter genes in unique, tissue-specific patterns. Of special interest is the identification of neural enhancer traps that will be of great value in future studies on brain development and behavior.